PROJECT SUMMARY Nucleolin recognition of MYC promoter G-quadruplex and its role in MYC regulation by MycG4-ligands G-quadruplexes (G4s) are non-canonical DNA secondary structures. c-Myc, one of the central driver oncogenes in human cancers, has a DNA G4 motif in its proximal promoter region that functions as a transcription silencer. However, little is known about how the c-Myc promoter G4 (MycG4) is regulated. Nucleolin is identified as the major c-Myc G4 binding protein and shows a remarkably higher binding affinity for MycG4 over its known cellular substrate NRE RNA. Nucleolin directly binds to the MycG4 promoter region in vivo and overexpression of nucleolin represses the activity of the c-Myc promoter. We hypothesize that nucleolin recognizes the c-Myc promoter G4 and that MycG4-targeted small molecules regulate c-Myc gene expression through interactions with the nucleolin-MycG4 complex. In this proposal, we will study how nucleolin recognizes MycG4 by determining the molecular structure of the nucleolin-MycG4 complex. We will also study how small molecules interact with the nucleolin-MycG4 complex and how the interactions affect c-Myc transcription. The c-Myc promoter G4 is an attractive anticancer drug target. Our preliminary data show that the nucleolin-MycG4 complex is clearly involved in the c-Myc regulation by MycG4-ligands. The proposed research represents the first structural and functional study of the nucleolin-MycG4 complex. A structural level understanding of the nucleolin- MycG4 complex and its interactions with small molecules will provide important information for MycG4 function and MycG4-targeted drug development. The proposed research will use a combination of high-field NMR spectroscopy, X-ray crystallography, and other biophysical, biochemical, and cellular approaches. We have assembled a strong collaboration team. The specific aims are: 1) To determine the structure of nucleolin in complex with the c-Myc promoter G-quadruplex. Nucleolin is a multi-domain protein containing four tandem RNA- binding domains (RBDs). We hypothesize that nucleolin uses all four RBDs to wrap around MycG4 and recognize the G4 external loops to achieve a high-affinity binding. This structure would be the first to show how the unusual DNA G4 structure is recognized by a modular protein, likely through multi-valent interactions. 2) To investigate how small molecules interact with the nucleolin-MycG4 complex and their effects on c-Myc gene regulation. Our preliminary data shows that only MycG4-ligands that stabilize the nucleolin-MycG4 complex can lower c-Myc levels. We will use a combination of in vitro and cell-based assays to identify compounds that can stabilize the nucleolin-MycG4 complex and downregulate c-Myc. We will determine the specificity for the c-Myc gene and determine major cellular response for the top compounds. For the MycG4-ligands that form the stable ternary complexes with nucleolin-MycG4, we will also determine the structural basis of the nucleolin-MycG4 stabilization.